Laser Capture Microdissection of Archival Kidney Tissue for qRT-PCR

Fig. 1

Methyl Carnoy’s-fixed paraffin-embedded tissue section after LCM of glomeruli (black asterisk) and proximal tubules (double black asterisk). In each case demarcation of individual glomeruli and tubules with the cutting laser can clearly be seen. The white asterisk shows examples where outlined tubules have not been fully captured. This indicates the importance of using multiple overlapping laser spots to fully fuse the section to the polymer cap. Scale bar = 100 μm

Select cut and capture.

Create additional capture groups.

Cut and capture again as above.

10.

Offload caps and slides.

3.3 Extraction, Isolation, and Purification of Total RNA from LCM of Archival Tissue Samples

Molecular analysis of archival fixed paraffin-embedded LCM tissue presents a significant technical challenge to the researcher. As already noted, RNA is highly susceptible to fragmentation (strand breaks) and chemical modification during the fixation and paraffin embedding preservation process. The quality of the specimen, duration of preservation prior to LCM and the expediency of microdissection and subsequent processing also all impinge greatly upon the quantity and quality of the final RNA yield. The following procedures are based around the use of proprietary Arcturus kit reagents and standardized protocols that are designed to maximize the recovery of RNA of sufficient quality from LCM samples to permit downstream gene expression analysis.

3.3.1 Extracting Captured Tissue from LCM Caps

Prepare extraction tubes prior to commencing LCM. Reconstitute Pro K Mix with 300 μL of Reconstitution Buffer (see Note 6 ). This provides sufficient mix for 12 extractions. For each cap, pipette 40 μL of the reconstituted Pro K Mix into a 0.5 mL microcentrifuge tube. Keep on ice until use.

Once regions of interest are captured, insert CapSure™ Macro LCM Cap into the microcentrifuge tube using the LCM Cap Insertion Tool, taking care not to touch the transfer film surface (see Note 7 ). Tap the microcentrifuge tube to ensure the extraction buffer is covering the CapSure™ Macro LCM Cap. Seal the assembly using Parafilm.

Incubate assembly for 16–18 h at 50 °C (see Note 8 ).

Centrifuge assembly at 1000 × g for 2 min to collect cell extract and proceed directly with the RNA isolation protocol as detailed below.

3.3.2 Isolation of Total RNA from Microdissected Tissue

RNA (e.g., glomerular RNA) may be extracted from the digested tissue using the Paradise PLUS Reagent System RNA Extraction/Isolation (Arcturus), which incorporates an on-column DNase digestion step to remove genomic DNA contamination. Briefly, the steps are as follows:

Pre-condition the RNA Purification Column:

(a)

Pipette 250 μL Conditioning Buffer (CB) onto the purification column filter membrane and incubate for 5 min at room temperature.

(b)

Centrifuge the purification column in a collection tube at 16,000 × g for 1 min.

Pipette 50 μL of 70 % Ethanol into the cell extract obtained from 3.3.1. Mix well by pipetting up and down. Do not centrifuge.

Pipette the cell extract and ethanol mixture (total volume ~80 μL) into the preconditioned column.

Centrifuge for 2 min at 100 × g to bind RNA to Column membrane filter, immediately followed by a centrifugation at 16,000 × g for 30 s. Discard flow-through.

Pipette 100 μL Wash Buffer (W1) into the purification column and centrifuge for 1 min at 8000 × g. Discard flow-through (see Note 9 ).

Prepare DNase incubation mix by adding 2 μL DNase I Mix to 20 μL DNase Buffer. Mix by gently inverting—do not vortex.

Pipette the Complete DNase incubation mix directly into the center of the column membrane. Incubate at room temperature for 25 min (see Note 10 ).

Pipette 40 μL RNA Kit Wash Buffer 1 (W1) into the purification column membrane. Centrifuge at 8000 × g for 15 s.

Pipette 100 μL Wash Buffer 2 (W2) into the purification column and centrifuge for 1 min at 8000 × g.

10.

Pipette another 100 μL Wash Buffer (W2) into the purification column and centrifuge for 2 min at 16,000 × g. Check the purification column for any residual wash buffer. If wash buffer remains recentrifuge at 16,000 × g for 1 min and discard any flow-though.

11.

Transfer the purification column to a new 0.5 mL microcentrifuge tube.

12.

Pipette 12 μL Elution Buffer (EB) directly onto the membrane of the purification column. Gently touch the tip of the pipette to the surface of the membrane while dispensing the elution buffer to ensure maximum absorption of EB into the membrane filter.

13.

Incubate the purification column for 1 min at room temperature.

14.

Centrifuge the column for 1 min at 1000 × g to distribute EB in the column, then for 1 min at 16,000 × g to elute total RNA. Optional: Retain 1 μL of sample to determine RNA yield (see Note 11 ). The remaining sample may be used immediately or stored at −80 °C until use.

3.4 cDNA Synthesis, Purification, and Amplification

The RNA yield from LCM is typically very low (<5–200 ng). Since several micrograms of RNA are needed for array-based applications, the RNA must be amplified. Conventional in vitro transcription (IVT) methods rely on the presence of an intact 3′ polyA tail for amplification by T7 RNA polymerase. However, RNA derived from paraffin-embedded fixed tissue is often degraded, which may lead to preferential amplification of transcripts with intact 3′ ends and thus biased expression analyses. As described here, Whole Transcriptome Amplification (WTA) reagents, such as those employed by the Paradise^® PLUS Reagent (Arcturus) system, circumvent the issue of 3′ end bias by the use of unique chimeric DNA–RNA primers. The DNA portion of these oligonucleotides hybridizes randomly across the entire transcriptome, while the RNA portion encodes a sequence that serves as a priming site for linear amplification by T7 RNA polymerase. Reverse transcription proceeds from the 3′ DNA end of each primer generating first strand cDNA. Second strand synthesis is then directed by the addition of a second exogenous primer, which generates dsDNA. Column-based purification of this cDNA followed by IVT with T7 RNA polymerase yields antisense RNA (aRNA) before isolation and a further round of amplification (optional for some applications). Detailed protocol notes can be found in the manual for the Paradise PLUS reagent system but the key steps and suggested modifications are given below.

3.4.1 Round One: First Strand cDNA Synthesis

Add 1.0 μL of Primer 1 (Gray-1) to 10–11 μL of purified RNA (input >20 ng) and incubate mixture for 1 h at 70 °C in a thermal cycler.

Cool samples to 4 °C for at least 1 min.

Prepare First Strand Synthesis Reaction Mix by combining 2 μL of Enhancer (Yellow), 5 μL of first Strand Master Mix (Red-1), 1 μL of first Strand Enzyme Mix (Red-2) and 1 μL of SuperScript™ III RT Enzyme (Invitrogen). Scale up with 10 % overage for multiple samples.

Add 9 μL of this complete First Strand Synthesis Reaction Mix to each sample, mix thoroughly and incubate at 42 °C for 1.5 h.

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